Revised Abstract

1. 606 Bacteriophage Therapy of Staphylococcus Aureus Including MRSA Sarah J Kuhl MD, PhD and Greg KearneyResearch and Medicine Services, VA Northern California Health Care System (VANCHCS), Martinez, CA Poland: Wroclaw Institute 2000 Approximately 0.1 cc of each overnight culture was mixed with 3-5 cc 0.7% tryptic soy top agar that had been melted and cooled to 45 degrees centigrade. This was immediately poured onto fresh plates of trypticsoy 1.5% agar, and allowed to solidify. Five or ten microliters of each phage dilution was spotted onto the top agar, and allowed to dry. The plates were then incubated overnight at 37 degrees centigrade. Plaques were counted at each dilution and titers were expressed as plaque-forming units of the original phage solution. Dilutions lower than 1/500 were not tested. Revised Abstract Furthermore, staphylococcus remained one of the major indications for therapeutic bacteriophage requests to the Bacteriophage Service of the Pasteur Institute (IP) in Paris (Vieu, 1961). More than half of the requests to the IP for adapted phage were for staphylococcal infections. Most of the staphylococcal strains were resistant in vitro to multiple antibiotics. The Pasteur Institutes of Paris and Lyon made adapted phage until the mid 1990s. Weber-Dabrowska et al (2000): 1307 patients with suppurative bacterial infections caused by multidrug-resistant bacteria were treated with specific bacteriophages. The infections were caused by Staphylococcus aureus, Escherichia coli, Klebsiella, Proteus, and Pseudomonas. Full recovery was noted in 1123 cases (85.9%).Transient improvement was observed in 134 cases (10.9%), and only in 50 cases (3.8%) was bacteriophage therapy found to be ineffective. Phage effectively treated bacterial infections resistant to available antibiotics. Background: Phage therapy once competed successfully with antibiotics in the treatment of human bacterial infections. Phage therapy was first used in France in 1919, and is still used in Eastern and Western Europe. Phage therapy was successfully used in the US in the preantibiotic era, and has an important potential role in the treatment of antibiotic-resistant infections. Phage therapy improved the mortality of Staphylococcus aureus bacteremia in over 500 cases treated in New York 1931-40 by MacNeal. The majority of phages produced for specific infections by the Pasteur Institute of Paris from 1959-1960 were for staphylococcal infections resistant to multiple antibiotics. Phage therapy continues to be used in the former Soviet Union, in Poland, and is undergoing a renaissance in Belgium, France, and Australia.   Methods:  14 clinical isolates of S. aureus were obtained from the microbiology laboratory at VA Northern California. Therapeutic preparations of Pyophage used in the Republic of Georgia were obtained from Eliava Institute (Zemphira Alavidze), and from Biochimpharm. The polyvalent bacteriophage of Gratia is manufactured in the United States for veterinary use as Staphage Lysate (SPL) by Delmont Labs. Phage were tested for activity against the  sequenced USA 300 MRSA strain BAA-1556 obtained from ATCC.  Results:  The MRSA strain BAA-1556 (USA 300) strain was susceptible to two bacteriophage cocktails from the Republic of Georgia, and to staphage lysate (SPL). Three of four MRSA clinical isolates were susceptible to all three phage preparations. Six of ten MSSA clinical isolates were susceptible to all three phage preparations. Each strain was tested several times. Conclusion: The epidemic USA 300 MRSA strain is susceptible to two commercial bacteriophage preparations from the Republic of Georgia, and to SPL produced in the US. Current studies support the use of phage therapy as practiced in France and Georgia, and in the preantibiotic era in the US. Phage treatment merits more aggressive investigation as an alternative to antibiotics as bacteria become increasing antibiotic resistant. Results USA The epidemic USA300 strain BAA1556, and most of the MRSA strains were sensitive to more than one phage widely used in human phage therapy D’Herelle gave the “Lane Lectures” at Stanford in the fall of 1928, and these were incorporated into his book, “The Bacteriophage and its Clinical Application” He was a professor at Yale from 1928-1933. There continued to be several sites of phage production and use, for example, the “polyvalent bacteriophage of Gratia was produced in the 1930s by the Michigan Department of Health, and later as Staphage lysate. MacNeal et al (1942) reported a decrease in mortality with bacteriophage treatment of staphylococcal bacteremia: Conclusions Background Antibiotic therapy may be entering a catastrophic or cataclysmic conclusion with no clear alternatives in place. Phage therapy has a long history of successful use, and merits funding and research. Bacteriophage have been used to treat human infections for more than 9 decades. Selected highlights follow: Georgia & Former Soviet Union Selected References D’Herelle and Eliava met at Institute Pasteur in Paris. Eliava returned to Georgia and founded what is now the Eliava Institute in the 1920s. He brought d’Herelle to Tbilisi in1933 and d’Herelle spent his time between Tbilisi and Paris until Eliava was murdered in 1937. The Institute became the major site of phage research and production for the former Soviet Union. The Institute has produced staphylococcal phage for both topical and intravenous use, and continues to be a major site for phage research and production. Phage are also made in Russia, the Ukraine, and other FSU countries. Belgium 1921 • Abedon, Kuhl, Blasdel, Kutter. 2011 Phage treatment of human infections. Bacteriophage 1:2, 66-85. • Kuhl and Mazure,2011, translation of d’Herelle’s “Preparation of a Therapeutic Bacteriophage, 1938. Bacteriophage 1:2, 55-65. • Kutter, Kuhl, Alavidze, Blasdel.l2011 Phage therapy. Textbook of Natural Medicine, in press. • Kutter, DeVos, Gvasalia, Alavidze, Gogokhia, Kuhl, Abedon. 2010. Phage therapy in clinical practice: treatment of human infections. Curr Pharm Biotechnol. 11(1):69-86. Therapeutic trials using bacteriophage of Staphylococcus by Bruynoghe and Maisin (in French), 1921 Phage were injected in 6 patients with pustules or boils, as close as possible to the diseased area. Lesions expansion stopped almost immediately, and completely disappearance in 24 to 48 hours Side effects were mild and included low grade fever in some patients. France Methods Acknowledgements D’Herelle codiscovered bacteriophage, and did the earliest human and animal phage therapy studies. D’Herelle’s laboratory made phage commercially from the 1930s until 1978 (see advertisement for Bacte-Staphy-Phage from the Laboratoire du Bacteriophage in a French medical journal in 1961). Staphylococcus aureus strains were obtained from the clinical microbiology lab at VANCHCS. The epidemic S. aureus USA300 strain BAA 1556 was obtained from ATCC. Strains were cultured overnight in Bacto Tryptic Soy broth. Phage obtained from the Republic of Georgia (Eliava Institute and Biochimpharm) and staphagelystae were titered by standard methods (Karam et al, 1994) . Phage dilutions were done in tryptic soy broth. • PhageBiotics Foundationfunded this study. • Elizabeth Betty Martin Kutter PhD • Zemphira Alavidze PhD • Guram Gvasalia MD, PhD • Chris Smith